Fig. 2.

Caffeine has convergently evolved in five flowering plant species using different combinations of genes and pathways. (A) Phylogenetic relationships among orders of Rosids and Asterids show multiple origins of caffeine biosynthesis. Lime-green lineages trace the ancient CS lineage of enzymes that has been independently recruited for use in caffeine-accumulating tissues in Theobroma, Paullinia, and Camellia. Turquoise lineages trace the ancient XMT lineage that was independently recruited in Citrus and Coffea. (B and C) Theobroma and Paullinia have converged upon similar biosynthetic pathways catalyzed by CS-type enzymes. (D) Citrus has evolved a different pathway catalyzed by XMT-type enzymes, despite its close relationship to Paullinia. (E and F) Camellia and Coffea catalyze the same pathway using different enzymes. Proposed biochemical pathways are based on relative enzyme activities shown by corresponding bar charts that indicate mean relative activities (from 0 to 1) with eight xanthine alkaloid substrates. CisXMT1 and TCS1 catalyze more than one reaction in the proposed pathways. XMT and CS have recently and independently duplicated in each of the five lineages (see Fig. S1 for a detailed gene tree). ^#Data taken from the literature; *substrate not assayed.